Liposomes containing a cisplatin compound

ABSTRACT

A liposome composition containing an entrapped cisplatin compound is described. The liposomes have a surface coating of hydrophilic polymer chains on inner and outer surfaces and an entrapped cisplatin compound. The compound is entrapped with substantially greater retention in the liposomes, when compared to liposomes lacking the polymer coating. A method of preparing the composition is also described.

This application claims the priority of U.S. Provisional ApplicationSerial No. 60/024,350, filed Aug. 23, 1996, which is incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates to a liposomal composition containing anentrapped cisplatin compound.

References

Freise, J., et al., Arch. Int. Pharmacodyn. 258:180-192 (1982).

Gondal, J. A., et al., Eur. J. Cancer 29A (11):1536-1542 (1993).

Mabrey, S., et al., Biochem. 17:2464-2468 (1978).

Martin, F. J., in SPECIALIZED DRUG DELIVERY SYSTEMS-MANUFACTURING ANDPRODUCTION TECHNOLOGY, (P. Tyle, Ed.) Marcel Dekker, New York, pp.267-316 (1990).

PHYSICIAN'S DESK REFERENCE, 48TH EDITION, Medical Economics DataProduction Co., Montvale, N.J. (1994).

Potkul, R. K., et al., Am. J. Obstet Gynecol. 164 (2): 652-658 (1991).

Prestayko, A. W., CANCER AND CHEMO. VOL III (Crooke, et al., Eds.)Academic Press, New York, 133-154 (1981).

Steerenberg, P. A., et al, International Journal of Pharmaceutics40:51-62 (1987).

Sur, B., et al, Oncology 40:372-376 (1983).

Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980).

Tsong, T. Y., Biochem. 14:5409-5414, 5415-5417 (1975).

Weiss, R. B., et al., Drugs 46(3):360-377 (1993).

BACKGROUND OF THE INVENTION

Cisplatin--cis-diamine-dichloroplatinum (II)--is one of the moreeffective anti-tumor agents used in the systemic treatment of germ cellcancers. This chemotherapeutic drug is highly effective in the treatmentof tumor models in laboratory animals and in human tumors, such asendometrial, bladder, ovarian and testicular neoplasms, as well assquamous cell carcinoma of the head and neck (Sur, et al., 1983;Steerenberg, et al., 1987).

Like other cancer chemotherapeutic agents, cisplatin is a highly toxicdrug. The main disadvantages of cisplatin are its extremenephrotoxicity, which is the main dose-limiting factor, its rapidexcretion via the kidneys, with a circulation half life of only a fewminutes, and its strong affinity to plasma proteins (Freise, et al.,1982).

Attempts to minimize the toxicity of the drug have included combinationchemotherapy, synthesis of cisplatin analogues (Prestayko, 1991; Weiss,et al., 1993), immunotherapy and entrapment in liposomes (Sur, et al.,1983; Weiss, et al., 1993). Antineoplastic agents, including cisplatin,entrapped in liposomes have a reduced toxicity, relative to the agent infree form, while retaining antitumor activity (Steerenberg, et al.,1987; Weiss, et al., 1993).

Cisplatin, however, is difficult to efficiently entrap in liposomesbecause of the drug's low aqueous solubility, approximately 1.0 mg/ml atroom temperature, and low lipophilicity, both of which contribute to alow drug/lipid ratio.

Liposomes containing cisplatin suffer from another problem--stability ofthe composition. In particular, maintenance of drug potency andretention of the drug in the liposome during storage are recognizedproblems (Freise, et al., 1982; Gondal, et al., 1993; Potkul, et al.,1991; Steerenberg, et al., 1987; Weiss, et al., 1993) and a limitedshelf life of liposomes containing cisplatin, on the order of severalweeks at 4° C., has been reported (Gondal, et al., 1993; Potkul, et al.,1991).

SUMMARY OF THE INVENTION

In one aspect, the invention includes a liposomal composition containingan entrapped cisplatin compound. The composition includes liposomeshaving an outer surface and an inner surface defining an aqueousliposome compartment. The liposomes are composed of a vesicle-forminglipid and between about 1-20 mole percent of a vesicle-forming lipidderivatized with a hydrophilic polymer. The liposomes are formed suchthat the hydrophilic polymer forms a coating of hydrophilic polymerchains on both the inner and outer surfaces. The cisplatin compound isentrapped in the liposomes with substantially greater retention than inliposomes lacking the inner and outer polymer surface coatings.

The cisplatin compound, in one embodiment, is native cisplatin and isentrapped in the liposomes at a drug-to-lipid ratio of between about 10to 20 μg/mg total lipid. In another embodiment, the cisplatin compoundis a cisplatin analogue.

In another embodiment, the hydrophilic polymer chains are composed of ahydrophilic polymer selected from the group consisting ofpolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, and polyaspartamide. In a preferred embodiment, thehydrophilic polymer is polyethyleneglycol.

The liposomes, in one embodiment have sizes between about 80-160 nm,preferably 100-140 nm, most preferably between about 100-120 nm.

In a preferred embodiment, the vesicle forming lipid is hydrogenated soyphosphatidylcholine and the derivatized vesicle forming lipid isdistearyl phosphatidylethanolamine derivatized with polyethylene glycol.

In another aspect, the invention includes a method of entrapping acisplatin compound in liposomes, by heating an aqueous solution of acisplatin compound to a temperature sufficient to increase itssolubility over the compound's solubility at room temperature. To theheated cisplatin compound solution is added a vesicle-forming lipid andbetween about 1-20 mole percent of a vesicle-forming lipid derivatizedwith a hydrophilic polymer. By said adding, liposomes having an innersurface coating and an outer surface coating of hydrophilic polymerchains are formed and the cisplatin compound is entrapped in theliposomes with substantially greater retention than in liposomes lackingthe polymer coatings.

In one embodiment of the method, the cisplatin compound is nativecisplatin and the aqueous cisplatin solution is heated to a temperaturesufficient to achieve a two-fold to eight-fold increase in cisplatinsolubility over its room temperature solubility.

In another embodiment, a solution of vesicle-forming lipids heated towithin about 10° C. of the temperature of the cisplatin solution isadded to the cisplatin compound solution.

In another embodiment, added to the cisplatin solution is a solutioncontaining a vesicle-forming lipid having a phase transition temperaturewithin about 10° C. of the temperature to which the cisplatin solutionis heated.

In another embodiment, the vesicle-forming lipid solution added to thecisplatin solution contains a vesicle-forming lipid having a phasetransition temperature between 40-70° C.

These and other objects and features of the invention will be more fullyappreciated when the following detailed description of the invention isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a liposome formed in accordancewith the present invention;

FIG. 2 illustrates a general reaction scheme for derivatizing avesicle-forming lipid with a polyalkylether;

FIG. 3 is a reaction scheme for preparing phosphatidylethanolaminederivatized with polyethyleneglycol via a cyanuric chloride linkingagent;

FIG. 4 illustrates a reaction scheme for preparingphosphatidylethanolamine derivatized with polyethyleneglycol by means ofa diimidazole activating reagent;

FIG. 5 is a plot of tumor volume, in mm³, as a function of days postinoculation for mice inoculated with C26 colon tumor model and treatedwith on days 12, 19 and 26 with saline (▪), free cisplatin (▴), freecarboplatin () or with liposome-entrapped cisplatin (▾);

FIG. 6 is a plot of tumor volume, in mm³, as a function of days postinoculation for mice inoculated with C26 colon tumor model and treatedwith on days 12, 19 and 26 with saline (▪), free cisplatin (▴), or withliposome-entrapped cisplatin according to three dosing regimens (▾, ♦,); and

FIG. 7 is a plot of tumor volume, in mm³, as a function of days postinoculation for mice inoculated with Lewis lung carcinoma and treatedwith on days 7, 14 and 21, with saline (▪), free cisplatin IV (♦) , freecisplatin IP () or with liposome-entrapped cisplatin IV (▴) or IP (▾).

DETAILED DESCRIPTION OF THE INVENTION I. Liposome Composition

The liposomal composition of the present invention includes liposomeshaving a stably entrapped cisplatin compound. As used herein, "stablyentrapped cisplatin compound" refers to native cisplatin or a cisplatinanalogue captured within a liposome, primarily within the aqueous spaceof the liposome, such that the cisplatin compound is retainedsubstantially within the liposome prior to administration.

FIG. 1 illustrates a liposome 10, prepared in accordance with theinvention, which includes an inner lipid bilayer 12 and an outer lipidbilayer 14. The inner and outer lipid bilayers are formed predominantlyof vesicle-forming lipids, such as lipid 16, which include a polar headgroup 16a and a hydrophobic tail 16b. Exemplary vesicle-forming lipidsare listed below.

Liposome 10 also includes vesicle-forming lipids derivatized with ahydrophilic polymer, such as derivatized lipid 18 in FIG. 1. Derivatizedlipid 18 includes a hydrophobic tail 18a, a polar head group 18b, andattached to the polar head group, by means described below, ahydrophilic polymer 18c. The hydrophilic polymer provides surfacecoatings 20, 21 of hydrophilic polymer chains on both an outer surface22 of outer lipid bilayer 14 and an inner surface 24 of inner lipidbilayer 12. The outer surface coating of hydrophilic polymer chains iseffective to provide a liposome with a long blood circulation lifetimein vivo. As will be illustrated below, the inner and outer surfacecoatings are further effective in providing a cisplatin liposomescomposition with a long shelf life, e.g., a stable liposome compositionwhere the cisplatin compound is retained in the liposome.

Liposome 10 also includes a cisplatin compound, e.g., native cisplatinor a cisplatin analogue, in entrapped form. The drug is entrapped in theinner aqueous compartment 26 in dissolved form or in precipitated form.In studies performed in support of the invention, native cisplatin wasentrapped in liposomes prepared with a surface coating of hydrophilicpolymer chains on the liposome's inner and outer surfaces. Theseliposomes, when compared to liposomes lacking the hydrophilic polymercoating, had an improved stability, as evidenced by the ability toretain the drug in its native state within the liposome for a longerperiod of time.

A. Vesicle-Forming Lipid Component

The liposome composition of the present invention is composed primarilyof vesicle-forming lipids. Such a vesicle-forming lipid is one which (a)can form spontaneously into bilayer vesicles in water, as exemplified bythe phospholipids, or (b) is stably incorporated into lipid bilayers,with its hydrophobic moiety in contact with the interior, hydrophobicregion of the bilayer membrane, and its polar head group moiety orientedtoward the exterior, polar surface of the membrane.

The vesicle-forming lipids of this type are preferably ones having twohydrocarbon chains, typically acyl chains, and a polar head group. Thereare a variety of synthetic vesicle-forming lipids andnaturally-occurring vesicle-forming lipids, including the phospholipids,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin(SM), where the two hydrocarbon chains are typically between about 14-22carbon atoms in length, and have varying degrees of unsaturation. Apreferred lipid for use in the present invention is hydrogenated soyphosphatidylcholine (HSPC).

The above-described lipids and phospholipids whose acyl chains havevarying degrees of saturation can be obtained commercially or preparedaccording to published methods.

The vesicle-forming lipid may be selected to achieve a specified degreeof fluidity or rigidity, to control the stability of the liposome inserum and to control the rate of release of the entrapped agent in theliposome. Liposomes having a more rigid lipid bilayer, or a liquidcrystalline bilayer, are achieved by incorporation of a relatively rigidlipid, e.g., a lipid having a relatively high phase transitiontemperature, e.g., up to 80° C. Rigid, i.e., saturated, lipidscontribute to greater membrane rigidity in the lipid bilayer. Otherlipid components, such as cholesterol, are also known to contribute tomembrane rigidity in lipid bilayer structures.

Lipid fluidity is achieved by incorporation of a relatively fluid lipid,typically one having a lipid phase with a relatively low liquid toliquid-crystalline phase transition temperature, e.g., at or below roomtemperature (20-25° C.).

Lipids suitable for use in the cisplatin liposome composition of thepresent invention include vesicle-forming lipids having phase transitiontemperatures at or below room temperature and those having a high phasetransition temperature. In a preferred embodiment, a vesicle-forminglipid having a phase transition temperature between about 40-70° C. isemployed. In another embodiment, the lipid used in forming the liposomesis one having a phase transition temperature within about 20° C., morepreferably 10° C., most preferably 5° C., of the temperature to whichthe solution containing the cisplatin compound is heated during liposomepreparation, as will be described. Phase transition temperatures oflipids are tabulated in a variety of sources, such as Avanti PolarLipids catalogue and Lipid Thermotropic Phase Transition Database(LIPIDAT, NIST Standard Reference Database 34).

The liposomes may include other lipids that can stabilize a vesicle orliposome composed predominantly of phospholipids. A frequently employedlipid for this purpose is cholesterol at between 25 to 40 mole percent.At between 0 to 20 mole percent cholesterol in a bilayer, separatedomains exist containing cholesterol and phospholipids and purephospholipid (Mabrey, et al., 1978). These bilayers show an increasedpermeability to water (Tsong, 1975). In one embodiment of the presentinvention, cholesterol is included in the liposome composition, as willbe described below.

In the method of the invention, liposomes containing a cisplatincompound are prepared by adding to a heated aqueous solution of acisplatin compound a mixture of vesicle-forming lipids containingbetween 1-20 mole percent of a vesicle-forming lipid derivatized with ahydrophilic polymer. The lipids are dissolved in a suitable lipidsolvent, such as ethanol, methanol, chloroform or mixtures thereof.

Prior to adding the lipids, the aqueous solution of cisplatin compoundis heated to a temperature sufficient to increase its solubility overthe room temperature solubility of the cisplatin compound. That is, thesolution is heated to a temperature that achieves at least about atwo-fold, preferably a four-fold, most preferably an eight-fold,increase in solubility over the aqueous room temperature solubility ofthe cisplatin compound. For example, cisplatin, having an aqueoussolubility of 1 mg/ml at 20-25° C. may be heated to about 63° C. toincrease the aqueous solubility to about 8.5 mg/ml. The solubility ofcisplatin and of cisplatin analogues can be readily determined by one ofskill in the art according to standard procedures.

In one embodiment of the invention, the solution containing thevesicle-forming lipid is heated to within about 20° C., more preferably10° C., most preferably 50° C., of the temperature of the cisplatinsolution.

In the example detailed below, the vesicle-forming lipid HSPC, thederivatized vesicle-forming lipid PEG-DSPE and cholesterol are dissolvedin ethanol heated to about 65° C., just above HSPC phase transitiontemperature's between about 52-60° C. An aqueous solution of nativecisplatin is heated to between 63-67° C. The solutions are mixedtogether to form liposomes containing the cisplatin compound inentrapped form.

The method of the invention achieves a high encapsulation of cisplatin,typically encapsulating between 10-20 μg drug/mg lipid, and providesliposomes having, in addition to the outer surface coating, an innersurface coating of hydrophilic polymer chains, with the cisplatincompound stably entrapped within the liposome.

B. Derivatized Vesicle-forming Lipid Component

As discussed above, the liposomes of the present invention have asurface coating of hydrophilic polymer chains on both the inner andouter lipid bilayer surfaces. The surface coating is provided byincluding in the liposome composition between about 1-20 mole percent ofa lipid derivatized with a hydrophilic polymer.

Hydrophilic polymers suitable for derivatization with a vesicle-forminglipid include polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropylmethacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, and polyaspartamide. The polymers may be employed ashomopolymers or as block or random copolymers.

A preferred hydrophilic polymer chain is polyethyleneglycol (PEG),preferably as a PEG chain having a molecular weight between 500-10,000daltons, more preferably between 1,000-5,000 daltons. Methoxy orethoxy-capped analogues of PEG are also preferred hydrophilic polymers,commercially available in a variety of polymer sizes, e.g., 120-20,000daltons.

Vesicle-forming lipids suitable for derivatization with a hydrophilicpolymer include any of those lipids listed above, and, in particularphospholipids, such as distearyl phosphatidylethanolamine (DSPE).Preparation of DSPE derivatized with PEG is described below (Example 1).

The derivatized lipid is incorporated into the liposome by including anamphipathic lipid derivatized with hydrophilic polymer withvesicle-forming lipids during formation of the lipid vesicles.

Once a liposome has been formed, the hydrophilic polymer chains providea surface coating of chains on both the inner and outer surfaces of thelipid bilayer, as discussed with respect to FIG. 1. Importantly, thesurface coating is effective to improve the stability of the liposomes,as will be described below. The surface coating is also effective toextend the blood circulation time of the liposomes in the absence ofsuch a coating. The extent of enhancement of blood circulation time ispreferably severalfold over that achieved in the absence of the polymercoating, as described in co-owned U.S. Pat. No. 5,013,556.

C. Other Liposome Components

The liposomes in the composition of the present invention may includeother components, such as targeting molecules, including antibodies,antibody fragments, or cell-surface recognition molecules, which areattached to the liposome by means of a hydrophilic polymer chains. Forexample, a vesicle-forming lipid is derivatized with a hydrophilicpolymer chain, as described above, and the hydrophilic polymer isend-functionalized for coupling antibodies to its functionalized end.The functionalized end group may be a hydrazide or hydrazine group whichis reactive toward aldehyde groups, although any of a number ofPEG-terminal reactive groups for coupling to antibodies may be used.Hydrazides can also be acylated by active esters orcarbodiimide-activated carboxyl groups. Acyl azide groups reactive asacylating species can be easily obtained from hydrazides and permitattachment of amino-containing molecules. The functionalized end groupmay also be 2-pyridyldithio-propionamide, for coupling an antibody orother molecule to the liposome through a disulfide linkage.

D. Cisplatin Compound

The liposome composition of the present invention is intended for use incancer therapy, more particularly in tumor therapy. A cisplatin compoundis entrapped within the liposomes and the liposome composition isadministered to the subject. As referred to herein, cisplatin compoundrefers to native cisplatin and its analogues. In a preferred embodiment,the cisplatin compound is native cisplatin and in another embodiment,the cisplatin compound is a cisplatin analogue, preferably a hydrophiliccisplatin analogue.

Native cisplatin, also referred to herein as cisplatin, is a heavy metalcomplex containing a central atom of platinum surrounded by two chlorideatoms and two ammonia molecules in the cis position. It is a yellowpowder with the molecular formula PtCl₂ H₆ N₂, and a molecular weight of300.1. It is soluble at room temperature in water or saline at 1 mg/mland has a melting point of 207° C. (PHYSICIAN'S DESK REFERENCE, 1994)and decomposes at 270° C.

The chlorine atoms in the cisplatin molecule are subject to chemicaldisplacement reactions by nucleophiles, such as water or sulfhydrylgroups. In aqueous media, water molecules are potential ligands, whichmay replace the chlorine atoms to form monohydroxymonochloro cis-diamineplatinum (II).

The drug is available as a sterile aqueous solution containing 1 mgcisplatin and 9 mg NaCl per ml water and in this form is typicallyadministered intravenously for tumor therapy at a dose of between about20-120 mg/m² (PHYSICIAN'S DESK REFERENCE, 1994). The drug may beadministered alone or in combination with other chemotherapeutic agents,as a bolus injection or as a slow infusion over a period of severalhours.

As a single agent, cisplatin can be administered, for example, at a doseof 100 mg/m² intravenously once every 4 weeks (PHYSICIAN'S DESKREFERENCE, 1994) or at a dose of 20 mg/M² cisplatin given as a rapidintravenous infusion daily for 5 days and repeated at 4-week intervals(Prestayko, 1991). In treatment of squamous cell cancer of the head andneck, cisplatin given intravenously as a 24-hour infusion at a dose of80 mg/m² achieved favorable response (Prestayko, 1991).

While active as a single agent, cisplatin is often administered incombination with other agents, including vinblastine, bleomycin,actinomycin, adriamycin, prednisone, vincristine, and others (Prestayko,1991). For example, therapy of ovarian cancer may include 60 mg/m²cisplatin and 60 mg/m² adriamycin administered as a 24-hour infusion. Inthe present invention, combination therapy may includeliposome-entrapped cisplatin administered with another chemotherapeuticagent in free form or in liposome-entrapped form, such asliposome-entrapped doxorubicin described in U.S. Pat. No. 5,527,528.

In the present invention, cisplatin is entrapped in the liposomes whichare sized to between about 80-160 nm, more preferably between about100-140 nm, most preferably between about 100-120 nm, suitable forintravenous administration. The cisplatin-containing liposomes areadministered at the dosages given above for the free drug, however, suchdosages may be adjusted accordingly to account for the reduced toxicityof the drug provided by liposomal entrapment. Such an adjusted dosagecan be readily determined experimentally by one of skill in the art.

The internal liposomal concentration of cisplatin is between 1-9 mgcisplatin/ml, more preferably between 4-9 mg/ml, most preferably between6-8.5 mg/ml. The cisplatin-containing liposome suspension is at aconcentration of between about 1-2 mg cisplatin/ml total suspensionvolume, for intravenous administration.

In another embodiment of the invention, the cisplatin compound entrappedwithin the liposomes is a cisplatin analogue. A wide spectrum ofcisplatin analogues have been synthesized, offering a differentantitumor spectrum, better therapeutic index and reduced toxicity thanthat offered by native cisplatin. Such analogues include carboplatin,ormaplatin, oxaliplatin, DWA2114R((-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane dicarboxylato)platinum), zeniplatin, enloplatin,lobaplatin, CI-973 (SP-4-3(R)-1,1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N')platinum),254-S nedaplatin and JM-216(bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV)) (Weiss, etal., 1993). Some cisplatin analogues, such as spiroplatin, have beenfound to be more toxic than native cisplatin. While more toxic analoguesare not desirable for intravenous administration in free form, suchanalogues may have use in liposome-entrapped form, which reduces drugtoxicity.

For purposes of the present invention, analogues having some degree ofwater solubility, such as carboplatin, iproplatin and others, arepreferred so that the drug is entrapped primarily in the inner aqueouscompartment of the liposome.

In a preferred embodiment, the cisplatin analogue is carboplatin,(1,1-cyclobutane-dicarboxylate-diammineplatinum), which contains organicligands in a 4-coordinate planar complex of platinum. This cisplatinanalogue demonstrated equivalent or greater antitumor activity thancisplatin and less nephrotoxicity in preclinical studies (Weiss, et al.,1993).

Liposomes containing a cisplatin analogue may be administered alone orin combination with other chemotherapeutic agents, in free form or inliposome-entrapped form, as discussed above with respect to nativecisplatin.

II. Preparing the Liposome Composition

Section A below describes synthesis of vesicle-forming lipidsderivatized with a hydrophilic polymer for use in forming the liposomesof the present invention. Section B describes a method of preparingliposomes including the derivatized lipids and a cisplatin compound.

A. Preparation of Derivatized Vesicle-Forming Lipids

FIG. 2 shows a general reaction scheme for preparing a vesicle-forminglipid derivatized with a biocompatible, hydrophilic polymer, asexemplified by polyethylene glycol (PEG), which is readily watersoluble, can be coupled to vesicle-forming lipids, and is tolerated invivo without toxic effects. The polymer is preferably capped by amethoxy, ethoxy or other unreactive group at one end, or is a polymer inwhich one end is more reactive than the other.

The polymer is activated at one end by reaction with a suitableactivating agent, such as cyanuric acid, diimadozle, anhydride reagent,or the like, as described below. The activated compound is then reactedwith a vesicle-forming lipid, such as phosphatidylethanol (PE), toproduce the derivatized lipid.

Alternatively, the polar group in the vesicle-forming lipid may beactivated for reaction with the polymer, or the two groups may be joinedin a concerted coupling reaction, according to known coupling methods.PEG capped at one end with a methoxy or ethoxy group can be obtainedcommercially in a variety of polymer sizes, e.g., 500-20,000 daltonmolecular weights.

The vesicle-forming lipid is preferably one having two hydrocarbonchains, typically acyl chains, and a polar head group, such as thoselisted above.

FIG. 3 shows a reaction scheme for producing a PE-PEG lipid in which thePEG is derivatized to PE through a cyanuric chloride group. Details ofthe reaction are provided in Example 1. Briefly, methoxy-capped PEG isactivated with cyanuric chloride in the presence of sodium carbonateunder conditions which produced the activated PEG compound in thefigure. This material is purified to remove unreacted cyanuric acid. Theactivated PEG compound is reacted with PE in the presence oftriethylamine to produce the desired PE-PEG compound, also shown in thefigure. The yield is about 8-10% with respect to initial quantities ofPEG.

The method just described may be applied to a variety of lipid amines,including PE, cholesteryl amine, and glycolipids with sugar-aminegroups.

A second method of coupling a polyalkylether, such as capped PEG to alipid amine is illustrated in FIG. 4. Here the capped PEG is activatedwith a carbonyl diimidazole coupling reagent, to form the activatedimidazole compound shown in FIG. 4. Reaction with a lipid amine, such asPE leads to PEG coupling to the lipid through an amide linkage, asillustrated in the PEG-PE compound shown in the figure. Details of thereaction are given in Example 2.

B. Liposome Preparation

The liposomes may be prepared by a variety of techniques, such as thosedetailed in Szoka et al, 1980. One method for preparing drug-containingliposomes is the reverse phase evaporation method, described by Szokaand in U.S. Pat. No. 4,235,871. Reverse phase evaporation vesicles(REVs) have typical average sizes between 2-4 microns and arepredominantly oligolamellar, that is, contain one or a few lipid bilayershells.

In another method, multilamellar vesicles (MLVs) can be formed by simplelipid-film hydration techniques. In this procedure, a mixture ofliposome-forming lipids of the type detailed above dissolved in asuitable organic solvent is evaporated in a vessel to form a thin film,which is then covered by an aqueous medium. The lipid film hydrates toform MLVs, typically with sizes between about 0.1 to 10 microns.

The liposomes are then sized, and one effective sizing method for REVsand MLVs involves extruding an aqueous suspension of the liposomesthrough a series of polycarbonate membranes having a selected uniformpore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1,or 0.2 microns. The pore size of the membrane corresponds roughly to thelargest sizes of liposomes produced by extrusion through that membrane,particularly where the preparation is extruded two or more times throughthe same membrane. Homogenization methods are also useful fordown-sizing liposomes to sizes of 100 nm or less (Martin, 1990).

In the present invention, the liposome composition is typically preparedwith between about 25-80 mole percent vesicle-forming lipids, 10-40 molepercent cholesterol, and 1-20 mole percent polymer-derivatized lipid.One exemplary liposome formulation includes hydrogenated soyphosphatidylcholine (HSPC) and cholesterol (Chol), in about a 1:1 molarratio, and between about 1-5 mole % of DSPE-PEG, added to form liposomeswith an inner and outer bilayer surface coating of PEG.

Generally, the cisplatin compound is incorporated into liposomes duringliposome formation by adding the solution of lipids to a solutioncontaining the drug, as will be described below.

In accordance with the method of the invention, and as described inExample 3, a lipid solution containing between 50-200 mg/ml of totallipids consisting of mPEG-DSPE, HSPC and cholesterol (molar ratio of50.6/44.3/5.1) was prepared by dissolving the lipids in warm (60-65°)ethanol. An aqueous solution of cisplatin (8.5 mg/ml cisplatin in 0.9%sodium chloride) was warmed to a temperature sufficient to significantlyincrease its solubility over the compound's solubility at roomtemperature, specifically, the cisplatin solution was heated to about63° C., increasing the cisplatin solubility approximately eight-fold,from 1 mg/ml to 8.5 mg/ml.

The cisplatin solution and the lipid solution were added together toform liposomes, with the temperature of the mixture maintained atbetween 60-65° C.

The liposomes, following diafiltration and dialysis, were extrudedthrough 0.2 μm and 0.1 μm polycarbonate filters to size the liposomes tobetween about 100-120 nm. The liposome suspension was cooled to roomtemperature, and the unentrapped, precipitated cisplatin was removed byfiltering and by diafiltration.

The final liposomes contained an internal phase of cisplatinencapsulated at a concentration of 8.5 mg/ml in 0.9% sodium chloride andan external phase of sucrose/sodium chloride solution. Prior topackaging for stability studies, described below, and/or prior toadministration, the liposome suspension was brought to a cisplatinconcentration of 1.05 mg/ml with a sucrose/sodium chloride/histidinesolution and the pH was adjusted to 6.5.

III. Stability of the Liposome Composition

The stability of liposomes prepared as described above (Example 3) wasevaluated by (i) analyzing the liposomal suspension for cisplatin andplatinum concentrations, (ii) determining percent of encapsulatedplatinum, (iii) measuring liposome size, and (iv) measuring the pH ofthe liposome suspension, each as a function of time and temperature.

As described in Example 4, cisplatin concentration was measured byassaying the liposome suspension for cisplatin concentration by highpressure liquid chromatography (HPLC). In this method, an organicsolvent is added to the liposome sample to disrupt the lipid bilayer,releasing the entrapped cisplatin, prior to assaying for cisplatinconcentration. The platinum concentration of the liposomal suspensionwas determined by atomic absorption. The percentage of encapsulatedplatinum was measured by separating the liposomes from unencapsulateddrug via size-exclusion chromatography and assaying both the liposomaland drug fractions for platinum content by atomic absorption. Liposomesize was determined by dynamic light scattering.

The results are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Stability of Cisplatin-Containing Liposomes                                                 Cisplatin                                                                              Pt    %       Liposome                                       Time    Conc.    Conc. Encapsulated                                                                          size                                     Temp. (month) (mg/ml)  (mg/ml)                                                                             Platinum                                                                              (nm)   pH                                ______________________________________                                        -40° C.                                                                      0       0.92     0.71  100     109    6.50                                    1       0.86     0.71  99      106    6.55                                    3       0.86     --    --      114    6.49                              -20° C.                                                                      1       0.85     0.71  100     111    6.56                                    3       0.86     --    --      117    6.49                                    18      1.00     0.69  --      117    6.29                              2-8° C.                                                                      1       0.85     0.71  100     109    6.61                                    3       0.87     0.68  100     109    6.48                                    6       0.90     0.73  99      110    6.54                                    18      0.86     0.71  99      109    6.30                              30° C.                                                                       1       0.70     0.71  98      107    6.43                                    3       0.55     0.68  93      107    6.18                              40° C.                                                                       0.5     0.65     0.71  96      110    6.36                                    1       0.53     0.65  91      106    6.25                              ______________________________________                                    

Liposomes stored at temperatures of -40° C. and -20° C. showed nomeasurable loss in the concentrations of cisplatin or platinum afterstorage for three months. Nor was a significant change observed inliposome size or suspension pH after the three month storage period. At-20° C. and after 18 months of storage, the cisplatin was retained inthe liposomes, as evidenced by no significant loss in either thecisplatin and platinum concentrations. The liposome composition was alsostable when stored at 2-8° C. for 18 months. As seen in Table 1, nomeasurable losses in the concentrations of cisplatin or platinum wereobserved. At the 18 month time point, the percentage of encapsulatedplatinum was 99%, indicating that all but 1% of the platinum wasretained in the liposomes. That the entrapped platinum is in the form ofcisplatin is evident from the cisplatin concentration, which does notdecrease over the 18 month storage period.

Under more aggressive storage conditions of 30° C. and 40° C., somedecrease in cisplatin and platinum concentrations was observed, and thepercentage of encapsulated platinum was 93% after 3 months at 30° C. and91% after 1 month at 40° C. Little change in liposome size was observed.

The data indicates that the liposome composition of the presentinvention is effective to retain the cisplatin in the liposomes in itsnative form, thereby providing a stable liposome composition. Thisstability is evidenced in particular by the 18 month time point at 2-8°C., where the concentration of cisplatin remained constant and 99% ofthe platinum was encapsulated in the liposomes.

The liposomes of the present invention, having an inner and an outersurface coating of hydrophilic polymer chains, were compared to"conventional" liposomes, that is, liposomes lacking the inner and outersurface coating of hydrophilic polymer chains.

As described in Example 5, cisplatin-containing liposomes were preparedin accordance with the present invention from HSPC/Chol/mPEG-DSPE in amolar ratio of 50.6/44.3/5.1. A comparative liposome composition wasprepared, which was identical to the liposomes of the present invention,except mPEG-DSPE was replaced with the same molar amount of distearylphosphatidyl glycerol (DSPG), which has the same hydrocarbon tail andthe same charge in the polar head group as mPEG-DSPE. The comparativeliposome composition, lacking the hydrophilic polymer, did not have asurface coating of hydrophilic polymer chains on either the inner orouter lipid bilayers.

In a first study (Example 5A), the liposome compositions were incubatedat 60° C. for 6 hours. After incubation, the cisplatin concentration ofthe liposomal suspension, the percentage of encapsulated platinum,liposome size and suspension pH were measured, according to theprocedures described above. The results, summarized in Table 2, showthat after the incubation period, the liposome composition of thepresent invention had a 24% (0.38 mg/ml to 0.29 mg/ml) decrease incisplatin concentration, whereas the cisplatin concentration of thecomparative liposomal suspension decreased by 44% (0.25 mg/ml to 0.14mg/ml). The percentage of encapsulated platinum of the liposomes of thepresent invention was 96% after incubation. The comparative liposomeshad a percent platinum encapsulation of 82%, indicating that 18% of theplatinum-containing species had leaked from the liposomes. In anembodiment of the invention, the liposome composition is characterizedby a percent of encapsulated platinum of above about 85%, morepreferably 90%, when stored at 60° C. for 6 hours.

                  TABLE 2                                                         ______________________________________                                        Stability of PEG-Coated Liposomes and                                         Comparative Liposomes at 60° C. for 6 Hours                                    Incubation  Cisplatin                                                                              %                                                        Temperature Conc.    Encapsulated                                                                          Size                                     Formulation                                                                           and Time    (mg/ml)  Platinum                                                                              (nm) pH                                  ______________________________________                                        HSPC/chol/                                                                            0           0.38     100     116  --                                  mPEG-   60° C.; 6 hours                                                                    0.29     96      117  --                                  DSPE                                                                          HSPC/chol/                                                                            0           0.25     100     149  6.53                                DSPG    60° C.; 6 hours                                                                    0.14     82      148  6.62                                (comparative                                                                  composition)                                                                  ______________________________________                                    

In a second study (Example 5B), liposomes having the compositionsdescribed above for the data presented in Table 2 were incubated at 40°C. for 2 weeks, and the results are shown in Table 3. In this study, theliposomes were diluted to a cisplatin concentration of 1.0 mg/ml in ahistidine/sucrose/sodium chloride diluent described in Example 3G. Afterthe incubation period, a 29% (from 0.75 to 0.53 mg/ml) decrease in thecisplatin concentration of the suspension containing liposomes having aPEG coating was observed. The percentage of encapsulated platinum was95%. This data indicates that a portion of the cisplatin has convertedto another molecular species, such as monohydroxymonochloro cis-diamineplatinum, discussed above, and that only about 5% of theplatinum-containing species leaked from the liposome after incubation at40° C. for 2 weeks.

                  TABLE 2                                                         ______________________________________                                        Stability of PEG-Coated Liposomes and                                         Comparative Liposomes at 40° C. for 2 Weeks                                    Incubation  Cisplatin                                                                              %                                                        Temperature Conc.    Encapsulated                                                                          Size                                     Formulation                                                                           and Time    (mg/ml)  Platinum                                                                              (nm) pH                                  ______________________________________                                        HSPC/chol/                                                                            0           0.75     100     108  6.62                                mPEG-   40° C.; 2 weeks                                                                    0.53     95      114  6.09                                DSPE                                                                          HSPC/chol/                                                                            0           0.51     100     146  6.53                                DSPG                                                                          (comparative                                                                          40° C.; 2 weeks                                                                    0        B1      137  5.84                                compositon)                                                                   ______________________________________                                    

The comparative liposome composition, after storage at 40° C. for 2weeks, had no measurable cisplatin remaining in the liposomes,suggesting that nearly all of the cisplatin had converted to anothermolecular species. 81% of the platinum-containing species wasencapsulated in the liposome--in other words, 19% of theplatinum-containing species had leaked from the liposomes. Clearly,liposomes prepared in accordance with the invention to have an inner andouter surface coating of hydrophilic polymer chains have a substantiallygreater retention of cisplatin than the comparative liposomes.

In another study, comparative liposomes were prepared as described aboveand stored at 2-8° C. for 2 months. The stability data for this study issummarized in Table 4 along with the data from Table 1 at the sametemperature for liposomes prepared according to the invention forcomparison.

                  TABLE 4                                                         ______________________________________                                        Stability of PEG-Coated Liposomes and Comparative                             Liposomes at 2-8° C. for 2 Months                                                       Cis-                                                                 Time     platin  Pt    %                                                      (months) Conc.   Conc. Encapsulated                                                                          size                                   Formulation                                                                           at 2-8° C.                                                                      (mg/ml) (mg/ml)                                                                             Platinum                                                                              (nm) pH                                ______________________________________                                        HSPC/chol/                                                                            0        0.92    0.71  100     109  6.5                               mPEG-   1        0.85    0.71  100     109  6.61                              DSPE    3        0.87    0.68  100     109  6.48                                      6        0.90    0.73  99      110  6.54                                      18       0.86    0.71  99      109  6.30                              HSPC/chol/                                                                            0        0.51    0.44  100     146  6.53                              DSPG    2        0.11    0.42  98      143  6.50                              (comparative                                                                  composition)                                                                  ______________________________________                                    

It is clear from the data shown in Table 4 that compared to liposomeslacking an inner and outer surface coating of hydrophilic polymerchains, the liposome composition of the present invention having such asurface coating is effective to reduce the loss of cisplatin from theliposomes. In particular, the liposome composition is effective toreduce conversion of cisplatin to other molecular species, as evidencedby comparing the cisplatin concentrations and the percentage ofencapsulated platinum for the two compositions.

In summary, the stability data presented in Tables 2, 3 and 4 indicatethat liposomes prepared in accordance with the present invention, havingan inner and outer surface coating of hydrophilic polymer chains,provide a stably entrapped, liposomal composition of cisplatin by (1)reducing the conversion of cisplatin to other molecular species, and (2)reducing the leakage of cisplatin (and other platinum-containingspecies) from the liposomes.

IV. In Vivo Administration

Cisplatin-containing liposomes prepared in accordance with the inventionwere administered to tumor-bearing mice and compared to free cisplatinand carboplatin for antitumor efficacy. As described in Example 6, micebearing the C26 color tumor model were treated with theliposome-entrapped cisplatin composition of the present invention, withfree cisplatin or with free carboplatin. Free cisplatin was administeredintravenously at the maximally tolerated dose of cisplatin, 6 mg/kg onceper week for 3 weeks. Free carboplatin was given at a clinicallyequivalent dose to cisplatin of 100 mg/kg, also administeredintravenously at the same frequency. Liposome-entrapped cisplatin wasadministered intravenously at the same dose and frequency as freecisplatin, 6 mg/kg once a week for 3 weeks.

The results are shown in FIG. 5, where tumor volume in mm³ is shown as afunction of days post inoculation. Treatment of the test groups wasinitiated when a palpable tumor mass of about 100 mm³ was evident, atabout day 12. As indicated by the solid triangles along the x-axis, thetest groups were treated on days 12, 19 and 26. The tumor in animalsreceiving saline (▪) continued to grow. The animals treated with freecisplatin (▴) or with free carboplatin () had a similar reduction intumor growth relative to the untreated animals. Animals treated withcisplatin entrapped in liposomes having a coating of polyethyleneglycolon the inner and outer surfaces of the liposomes (▾) had a significantreduction in tumor size relative to the animals treated with the freedrug.

Another study was conducted according to Example 6, where theliposome-entrapped cisplatin was administered according to differentdosing schedules. That is, liposome-entrapped cisplatin was administeredwith a larger loading dose followed by smaller doses at the second andthird weeks. The results are shown in FIG. 6, where test animalsreceiving saline (▪) and free cisplatin (▴) at 6 mg/kg once a week for 3weeks showed continual increase in tumor mass. The animals treated withliposome-entrapped cisplatin had significantly reduced tumor massescompared to the animals treated with saline or free cisplatin. Antitumorefficacy of the liposome-entrapped cisplatin was similar for dosingschedules of 6 mg/kg once a week for 3 weeks (), an initial dose of 12mg/kg with subsequent doses of 4 mg/kg on days 19 and 26 (♦) and aninitial dose of 14 mg/kg with subsequent doses of 4 mg/kg on days 19 and26 (▾).

Mice bearing the Lewis lung carcinoma tumor model were treated withcisplatin, administered intravenously and intraperitoneally, or withliposome-entrapped cisplatin, administered similarly. As described inExample 6, treatment was initiated when a tumor mass of about 100 mm³was observed in the test animals, with treatment occurring on days 7, 14and 21 after inoculation. A control group of tumor-bearing mice weretreated with saline.

The results are shown in FIG. 7, where tumor volume, in mm³, is plottedas a function of days post inoculation. Treatment is indicated by thesolid triangles along the x-axis on days 7, 14 and 21. The tumor in micetreated with saline (▪) grew continually through the test period. Theresponse in tumor mass to free cisplatin administered intravenously (♦)or intraperitoneally () at a dose of 6 mg/kg on the indicated treatmentdays was similar. Treatment with 12 mg/kg liposome-entrapped cisplatin,administered intravenously (▴) or intraperitoneally (▾) on the indicatedtreatment days had improved antitumor efficacy relative to the animalstreated with free cisplatin. Administration intravenously andintraperitoneally were equally effective.

From the foregoing, it can be appreciated how various features andobjects of the invention are met. The liposome composition of thepresent invention includes liposomes having an inner surface coating andan outer surface coating of hydrophilic polymer chains and an entrappedcisplatin compound. The studies performed in support of the inventiondemonstrate that the cisplatin is stably entrapped in the liposomes, asevidenced by retention of cisplatin and platinum after incubation forvarious times at several temperatures. Cisplatin stably entrapped inliposomes offers the advantages of reduced toxicity and of improvedefficacy, relative to the drug administered in free form, since afteradministration the drug remains entrapped in the liposome with littleleakage to the bloodstream. The outer surface coating of PEG chainsprovide a long blood circulation lifetime allowing the liposomes toreach a target site, such as a tumor.

V. EXAMPLES

The following examples are intended to illustrate, but not limit, thescope of the invention.

Materials: Cisplatin was obtained from W. C. Heraeus GmbH (Hanau,Germany). DSPE was purchased from Avanti Polar Lipids (Birmingham, Ala.)and methoxy-polyethyleneglycol (mPEG), MW 2000 dalton, was obtained fromFluka Chemie AG (Buchs, Switzerland). Cholesterol was obtained fromCroda, Inc., (New York , N.Y.). HSPC was made by Lipoid K. G.(Ludwigshafen, Germany) and mPEG-DSPE was made by Sygena, Inc.,(Liestal, Switzerland).

Example 1 Preparation of PEG-Derivatized DSPE linked by CyanuricChloride

A. Preparation of Activated PEG

2-0-Methoxypolyethylene glycol 1900-4,6-dichloro-1,3,5 triazinepreviously called activated PEG was prepared as described in J. Biol.Chem. 252:3582 (1977) with the following modifications.

Cyanuric chloride (5.5 g; 0.03 mol) was dissolved in 400 ml of anhydrousbenzene containing 10 g of anhydrous sodium carbonate, and PEG-1900 (19g; 0.01 mol) was added and the mixture was stirred overnight at roomtemperature. The solution was filtered, and 600 ml of petroleum ether(boiling range, 35-60°) was added slowly with stirring. The finelydivided precipitate was collected on a filter and redissolved in 400 mlof benzene. The precipitation and filtration process was repeatedseveral times until the petroleum ether was free of residual cyanuricchloride as determined by high pressure liquid chromatography on acolumn (250×3.2 mm) of 5-m "LiChrosorb" (E. Merck), developed withhexane, and detected with an ultraviolet detector. Titration ofactivated PEG-1900 with silver nitrate after overnight hydrolysis inaqueous buffer at pH 10.0, room temperature, gave a value of 1.7 mol ofchloride liberated/mol of PEG.

TLC analysis of the product was effected with TLC reversed-phase platesobtained from Baker using methanol: water, 4:1 (v/v) as developer andexposure to iodine vapor for visualization. Under these conditions, thestarting methoxy polyglycol 1900 appeared at R_(f) =0.54 to 0.60. Theactivated PEG appeared at R_(f) =0.41. Unreacted cyanuric chlorideappeared at R_(f) =8.88 and was removed.

The activated PEG was analyzed for nitrogen and an appropriatecorrection was applied in selecting the quantity of reactant to use infurther synthetic steps. Thus, when the product contained only 20% ofthe theoretical amount of nitrogen, the quantity of material used in thenext synthetic step was increased by 100/20, or 5-fold. When the productcontained 50% of the theoretical amount of nitrogen, only 100/50 or a2-fold increase was needed.

B. Preparation of N-(4-Chloro-polyglycol 1900)-1,3,5-Triazinyl EggPhosphatidylethanolamine

In a screw-capped test tube, 0.74 ml of a 100 mg/ml (0.100 mmole) stocksolution of egg phosphatidylethanolamine in chloroform was evaporated todryness under a stream of nitrogen and was added to the residue of theactivated PEG described in section A, in the amount to provide 205 mg(0.100 mmole). To this mixture, 5 ml anhydrous dimethyl formamide wasadded. 27 microliters (0.200 mmole) triethylamine was added to themixture, and the air was displaced with nitrogen gas. The mixture washeated overnight in a sand bath maintained at 110° C.

The mixture was then evaporated to dryness under vacuum and a pasty massof crystalline solid was obtained. This solid was dissolved in 5 ml of amixture of 4 volumes of acetone and 1 volume of acetic acid. Theresulting mixture was placed at the top of a 21 mm×240 mmchromatographic absorption column packed with silica gel (MerckKieselgel 60, 70-230 mesh) which had first been moistened with a solventcomposed of acetone acetic acid, 80/20; v/v.

The column chromatography was developed with the same solvent mixture,and separate 20 to 50 ml aliquots of eluent were collected. Each portionof eluent was assayed by TLC on silica gel coated plates, using2-butanone/acetic acid/water; 40/25/5; v/v/v as developer and iodinevapor exposure for visualization. Fractions containing only material ofR_(f) =about 0.79 were combined and evaporated to dryness under vacuum.Drying to constant weight under high vacuum afforded 86 mg (31.2micromoles) of nearly colorless solid N-(4-chloro-polyglycol1900)-1,3,5-triazinyl egg phosphatidylethanolamine containingphosphorous.

The solid compound was taken up in 24 ml of ethanol/chloroform; 50/50and centrifuged to remove insoluble material. Evaporation of theclarified solution to dryness under vacuum afforded 21 mg (7.62micromoles) of colorless solid.

Example 2 Preparation of PEG-Derivatized DSPE linked by Carbamate

A. Preparation of the Imidazole Carbamate of Polyethylene Glycol MethylEther 1900

9.5 grams (5 mmoles) of polyethylene glycol methyl ether 1900 obtainedfrom Aldrich Chemical Co. was dissolved in 45 ml benzene which had beendried over molecular sieves. 0.89 grams (5.5 mmoles) of pure carbonyldiimidazole was added. The purity was checked by an infra-red spectrum.The air in the reaction vessel was displaced with nitrogen. Vessel wasenclosed and heated in a sand bath at 75° C. for 16 hours.

The reaction mixture was cooled and a clear solution formed at roomtemperature. The solution was diluted to 50.0 ml with dry benzene andstored in the refrigerator as a 100 micromole/ml stock solution of theimidazole carbamate of PEG ether 1900.

B. Preparation of the Phosphatidylethanolamine Carbamate of PolyethyleneGlycol Methyl Ether 1900

10.0 ml (1 mmol) of the 100 mmol/ml stock solution of the imidazolecarbamate of polyethylene glycol methyl ether 1900 (compound X) waspipetted into a 10 ml pear-shaped flask. The solvent was removed undervacuum. 3.7 ml of a 100 mg/ml solution of egg phosphatidyl ethanolamine(V) in chloroform (0.5 mmol) was added. The solvent was evaporated undervacuum. 2 ml of 1,1,2,2-tetrachloroethylene and 139 microliters (1.0mmol) of triethylamine VI was added. The vessel was closed and heated ina sand bath maintained at 95° C. for 6 hours. At this time, thin-layerchromatography was performed with fractions of the above mixture todetermine the extent of conjugation on Si02 coated TLC plates, usingbutanone/acetic acid/water; 40/5/5; v/v/v; as developer. Iodine vaporvisualization revealed that most of the free phosphatidylethanolamine ofRf=0.68, had reacted, and was replaced by a phosphorous-containing lipidat R_(f) =0.78 to 0.80.

The solvent from the remaining reaction mixture was evaporated undervacuum. The residue was taken up in 10 ml methylene chloride and placedat the top of a 21 mm×270 mm chromatographic absorption column packedwith Merck Kieselgel 60 (70-230 mesh silica gel), which has been firstrinsed with methylene chloride. The mixture was passed through thecolumn, in sequence, using the following solvents.

    ______________________________________                                                  Volume % of  Volume % Methanol                                      ml        Methylene Chloride                                                                         with 2% Acetic Acid                                    ______________________________________                                        100       100%          0%                                                    200       95%           5%                                                    200       90%          10%                                                    200       85%          15%                                                    200       60%          40%                                                    ______________________________________                                    

50 ml portions of eluent were collected and each portion was assayed byTLC on Si02-coated plates, using I₂ vapor absorption for visualizationafter development with chloroform/methanol/water/concentrated ammoniumhydroxide; 130/70/8/0.5%; v/v/v/v. Most of the phosphates were found infractions 11, 12, 13 and 14.

These fractions were combined, evaporated to dryness under vacuum anddried in high vacuum to constant weight. They yielded 669 mg of acolorless wax of phosphatidyl ethanolamine carbamate of polyethyleneglycol methyl ether. This represented 263 micromoles and a yield of52.6% based on the phosphatidylethanolamine.

An NMR spectrum of the product dissolved in deutero-chloroform showedpeaks corresponding to the spectrum for egg PE, together with a strongsinglet due to the methylene groups of the ethylene oxide chain atDelta=3.4 ppm. The ratio of methylene protons from the ethylene oxide tothe terminal methyl protons of the PE acyl groups was large enough toconfirm a molecular weight of about 2000 for the polyethylene oxideportion of the molecule of the desired product polyethylene glycolconjugated phosphatidylethanolamine carbamate, M.W. 2,654.

Example 3 Liposome Preparation

A. Step 1: Drug Solution Preparation

Sterile water was heated to 63-67° C. in a TEFLON-lined pressure vesseland sodium chloride (0.9%) was added. Cisplatin was added at aconcentration of 8.5 mg/ml and mixed until dissolved, approximately15-25 minutes.

B. Step 2: Lipid Dissolution

257.0 g PEG-DSPE, 719.4 g HSPC and 308.4 g cholesterol (molar ratio of50.6/44.3/5.1) were added to 900 ml dehydrated ethanol at 60-65° C. andmixed until dissolved, approximately 2 hours. The dissolved lipids wereadded to 7670 g of drug solution to give a total lipid concentration ofapproximately 150 mg/ml.

C. Step 3: Lipid Hydration/Drug Loading

The warm lipid solution was rapidly added to the warm (63-67° C.) drugsolution, with mixing, to form a suspension of liposomes havingheterogeneous sizes. The suspension was mixed for one hour at 63-67° C.The cisplatin concentration in the hydration mixture was 7.2 mg/ml and,at this stage, approximately 30% of the drug was encapsulated in theliposomes. 10% of the total solution volume was ethanol and the totallipid concentration was 150 mg lipid/ml.

D. Step 4: Extrusion

The liposomes were sized to the desired mean particle diameter bycontrolled extrusion through polycarbonate filter cartridges housed inTeflon-lined stainless steel vessels. The liposome suspension wasmaintained at 63-65° C. throughout the extrusion process, a period of6-8 hours.

E. Step 5: Low-Grade Filtering

After sizing, the liposome suspension was cooled to room temperature(20-25° C.) and filtered through a 1.2 μm 142-mm Gelman Versapor filter(acrylic copolymer on a Nylon 66 support) to remove precipitated drug.At this stage approximately 50% of the drug was encapsulated.

F. Step 6: Diafiltration

A sucrose/sodium chloride solution was prepared by dissolving sucrose(100 mg/ml) and sodium chloride (0.058 mg/ml) in sterile water. The pHof the solution was adjusted to approximately 5.5 with 2N HCl or NaOH.The solution was filtered through a 0.22 μm Durapore filter.

The liposome suspension was diluted in approximately a 1:1 (v/v) ratiowith the sucrose/sodium chloride solution and diafiltered through apolysulfone hollow-fiber ultrafilter. Eight volume exchanges wereperformed against the sucrose/sodium chloride solution to remove theethanol and unencapsulated drug. The process fluid temperature wasmaintained at about 20-30° C. Total diafiltration time was approximately4.5 hours.

The liposome suspension was then concentrated to approximately 1.2 mgcisplatin/ml by ultrafiltration. The post diafiltration process fluidwas analyzed for cisplatin content by HPLC. The liposomes had aninternal phase of 8.5 mg/ml cisplatin in 0.9% sodium chloride and anexternal phase of sucrose/sodium chloride solution.

G. Step 7: Dilution

A diluent was prepared by dissolving histidine (10 mM) in asucrose/sodium chloride (10% sucrose/1 mM NaCl) solution to a targethistidine concentration of 1.55 mg/ml in the final mixture. The liposomesuspension was diluted to a target cisplatin concentration of 1.05 mg/mlwith the histidine diluent. The pH of the suspension was adjusted to 6.5with 2N NaOH or HCl.

H. Step 8: Sterile Filtration

The liposome suspension was heated to 33-38° C. and filtered through a0.2 μm Gelman Super polyethersulfone filter. Total filtration time wasapproximately 10 minutes.

Example 4 Stability of Cisplatin Liposomes

A suspension of cisplatin-containing liposomes was prepared as describedin Example 3. 5 ml aliquots of the liposome suspension were placed in 10cc glass vials, sealed under aseptic conditions, and placed inincubators or refrigerators at the following temperatures: -40° C., -20°C., 2-8° C., 30° C., 40° C. At intervals, samples from each vial storedat each temperature were drawn and tested in triplicate for:

1. Cisplatin concentration: The concentration of cisplatin was measuredby disrupting the liposome bilayer with an organic solvent to releasethe entrapped cisplatin and then determining the cisplatin concentrationby high pressure liquid chromatography (HPLC);

2. Platinum concentration, measured by atomic absorption;

3. Percent of encapsulated platinum: The percent encapsulated platinumwas determined by separating the liposomes from unencapsulated cisplatinby size-exclusion chromatography and assaying the liposomal and drugfractions for platinum content by atomic absorption;

4. Liposome size, determined by dynamic light scattering; and

5. pH of liposome suspension.

The results are shown in Table 1.

Example 5 Comparative Stability Studies

Cisplatin-containing liposomes were prepared with no inner and outersurface coating of hydrophilic polymer chains for comparison to theliposomes of the present invention. Comparative liposomes were preparedas described in Example 3, except distearyl phosphatidylglycerol (DSPG)was substituted for the PEG-DSPE derivative, e.g., the liposomecomposition consisted of HSPC/Chol/DSPG in a molar ratio of50.6/44.3/5.1.

A. Incubation at 60° C. for 6 Hours

Stability of the comparative liposome composition and the liposomecomposition of the present invention were compared by diluting theliposome samples with saline (1:1 v:v) and incubating the suspensionsfor 6 hours at 60° C. After incubation, the samples were tested forcisplatin concentration, % platinum encapsulation, liposome size and pH,according to the procedures described in Example 4. The results aresummarized in Table 2.

B. Incubation at 40° C. for 2 Weeks

The liposome compositions were diluted to a cisplatin concentration of 1mg/ml with the histidine/sucrose/ sodium chloride diluent described inExample 3G. The liposome suspensions were incubated at 40° C. for 2weeks, after which the cisplatin concentration, % platinumencapsulation, liposome size and pH were measured. The results aresummarized in Table 3.

Example 6 Treatment of Tumor-Bearing Mice

Liposomes containing entrapped cisplatin were prepared as described inExample 3 and were composed of HSPC, cholesterol and mPEG-DSPE in a50.6/44.3/5/1 molar ratio. The total lipid content was approximately 71mg/ml and the cisplatin concentration was 1 mg/ml.

A. C26 Colon Tumor Model

The C26 colon tumor model was grown in Balb/c, male mice (SimonsonLaboratories, Inc., Gilroy, Calif.). Tumors were harvested from donormice for and minced finely in RPMI media with 10% fetal calf serum(FCS). Tumors were then digested with enzyme mix (10 ml 0.25% proteasetype IX and 0.25% collagenase type IV in Hank's balanced salt solution(HBSS) and 0.2% ml 0.02% DNase in HBSS) for 30-60 minutes at 37° C.Single cell suspension of tumor cells was washed twice in RPMI mediawith FCS, concentrated by centrifugation and resuspended in RPMI mediawith FCS for inoculation. One million tumor cells (0.1 ml) wereinoculated in the left flank of 5-6 week old mice.

Throughout all experiments, animals were maintained in 12 hour light:12hour dark cycles, fed ad libitum rodent chow and water.

1. Comparison of Free Drug and Liposome-entrapped Drug. Thetumor-bearing mice were divided into groups of n=9 for treatment withliposome-entrapped cisplatin, free cisplatin (Platinol-AQ®, BristolLaboratories, Princeton, N.J.) or free carboplatin (Paraplatin®, BristolLaboratories) according to the treatment regimen shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                              Cumulative                                              Test Compound                                                                              IV Dose  Dose        Frequency                                   ______________________________________                                        Saline       0.1 ml   --          once/week × 3                         Cisplatin     6 mg/kg 18 mg       once/week × 3                         Carboplatin  100 mg/kg                                                                              300 mg      once/week × 3                         Liposomal Cisplatin                                                                         6 mg/kg 18 mg       once/week × 3                         ______________________________________                                    

Free cisplatin and liposome-entrapped cisplatin were administeredintravenously at a dose of 6 mg/kg weekly for 3 weeks. Carboplatin wasadministered intravenously at a dose of 100 mg/kg weekly for 3 weeks.The control group of mice (n=5) received 0.1 ml of saline intravenouslyweekly for 3 weeks. The results are shown in FIG. 5.

2. Altered Dosing Schedule. Mice were inoculated with the C26 colontumor cells according to the above-described procedure. Treatment of thetumor-bearing mice was initiated when the majority of animals hadpalpable tumors mass of about 100 mm³. The mice were treated withliposome-entrapped cisplatin or with free cisplatin once per week for 3weeks according to the regimen shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                                Cumulative                                            Test Compound                                                                             IV Dose     Dose      Frequency                                   ______________________________________                                        Saline      0.1 ml      --        once/week × 3                         Cisplatin   6 mg/kg     18 mg/kg  once/week × 3                         Liposomal Cisplatin                                                                       14/4/4 mg/kg.sup.1                                                                        22 mg/kg  once/week × 3                         Liposomal Cisplatin                                                                       12/4/4 mg/kg.sup.1                                                                        26 mg/kg  once/week × 3                         Liposomal Cisplatin                                                                       6 mg/kg     18 mg/kg  once/week × 3                         ______________________________________                                         .sup.1 Initial loading dose followed by 2 smaller doses.                 

Free cisplatin was administered intravenously at a dose of 6 mg/kg onceper week for 3 weeks to tumor-bearing mice (n=9). Cisplatin entrapped inliposomes, prepared as described above, was administered intravenouslyaccording to three dosing schedules to tumor-bearing mice. One group(n=9) received 6 mg/kg liposome-entrapped cisplatin once per week for 3weeks; another group (n=9) received a first dose of 14 mg/kg, followedby subsequent doses in weeks 2 and 3 of 4 mg/kg; another group (n=9)received a first dose of 12 mg/kg, followed by 4 mg/kg in weeks 2 and 3of the study. The control group (n=5) received saline on each treatmentday. The results are shown in FIG. 6.

B. Mice Bearing Lewis Lung Tumor Model

The Lewis lung tumor (LL/2, CRL-1642, ATCC, Rockville, Md.) model wasgrown in G6C3-F1, male mice (Simonson Laboratories, Inc., Gilroy,Calif.). Tumors were harvested from donor mice and processed as abovefor inoculation of an experiment. One million tumor cells (0.1 ml) wereinoculated in the left flank of 5-6 week old, male, B6C3-F1 mice.

Treatment of the tumor-bearing animals was initiated seven days afterinoculation, when the majority of animals had palpable tumor masses.Animals were treated according to the regimen shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                                Cumulative                                            Test Compound                                                                             Dose (Route)                                                                              Dose      Frequency                                   ______________________________________                                        Saline      0.1 ml (IV) --        once/week × 3                         Cisplatin   6 mg/kg (IV)                                                                              18 mg/kg  once/week × 3                         Cisplatin   6 mg/kg (IP)                                                                              18 mg/kg  ance/week × 3                         Liposomal Cisplatin                                                                       12 mg/kg (IV)                                                                             36 mg/kg  once/week × 3                         Liposomal Cispiatin                                                                       12 mg/kg (IP)                                                                             36 mg/kg  once/week × 3                         ______________________________________                                    

The control group (n=9) received saline on the treatment days (days 7,14 and 21). Two groups of animals received free cisplatin at 6 mg/kg oneach treatment day; in one group (n=9) the free cisplatin administeredintravenously, in the other group (n=9), the drug administeredintraperitoneally. Two groups received liposome-entrapped cisplatin at12 mg/kg on each treatment day; one group (n=7) receiving the doseintravenously, the other group (n=9) receiving the doseintraperitoneally. The results are shown in FIG. 7.

Although the invention has been described with respect to particularembodiments, it will be apparent to those skilled in the art thatvarious changes and modifications can be made without departing from theinvention.

It is claimed:
 1. A liposomal composition containing an entrappedcisplatin compound, comprisingliposomes having an outer surface and aninner surface defining an aqueous liposome compartment, and beingcomposed of a vesicle-forming lipid and between about 1-20 mole percentof a vesicle-forming lipid derivatized with a hydrophilic polymer havinga molecular weight between 1,000-5,000 Daltons and having an unchargedend cap, said liposomes being formed such that the hydrophilic polymerforms a coating of hydrophilic polymer chains on both said inner andouter surfaces, and the cisplatin compound entrapped in said liposomes,said compound remaining entrapped in the liposomes in its native formwith substantially greater retention when compared to liposomes lackingsaid polymer coating.
 2. The composition of claim 1, wherein saidhydrophilic polymer chains are composed of a hydrophilic polymerselected from the group consisting of polyvinylpyrrolidone,polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,polymethacrylamide, polydimethylacrylamide,polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, andpolyaspartamide.
 3. The composition of claim 2, wherein said hydrophilicpolymer chains are composed of polyethylene glycol.
 4. The compositionof claim 1, wherein said liposomes have sizes of between about 80-160nm.
 5. The composition of claim 1, wherein said vesicle forming lipid ishydrogenated soy phosphatidylcholine and said derivatized vesicleforming lipid is distearyl phosphatidylethanolamine derivatized withpolyethylene glycol.
 6. The composition of claim 1, wherein saidcisplatin compound is cisplatin or a cisplatin analogue selected fromthe group consisting of carboplatin, ormaplatin, oxaliplatin,((-)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutanedicarboxylato))platinum, zeniplatin, enloplatin, lobaplatin,(SP-4-3(R)-1,1-cyclobutane-dicarboxylato(2-)-(2-methyl-1,4-butanediamine-N,N'))platinum,nedaplatin and bis-acetato-ammine-dichloro-cyclohexylamine-platinum(IV).7. The composition of claim 6, wherein said cisplatin compound iscisplatin.
 8. The composition of claim 7, wherein said cisplatincompound is entrapped at a drug-to-lipid ratio of between about 10 to 20μg/mg total lipid.
 9. The composition of claim 1, wherein said liposomesare suspended in an aqueous medium at a liposome lipid concentrationbetween 50-200 mg/ml, and the composition is characterized by a percentof encapsulated platinum of above about 90% when stored at 60° C. for a6 hour period.
 10. A method of entrapping a cisplatin compound inliposomes, comprisingheating an aqueous solution of a cisplatin compoundto a temperature sufficient to increase its solubility at least two-foldover the compound's solubility at room temperature; adding to the heatedsolution a vesicle-forming lipid and between about 1-20 mole percent ofa vesicle-forming lipid derivatized with a hydrophilic polymer having amolecular weight between 1,000-5,000 Daltons and an uncharged end cap;and by said adding, forming liposomes having an inner surface coatingand an outer surface coating of said hydrophilic polymer and saidcisplatin compound remaining entrapped in the liposomes in its nativeform with substantially greater retention than in liposomes lacking saidinner and outer surface coating.
 11. The method of claim 10, whereinsaid heating includes heating native cisplatin to a temperaturesufficient to achieve a two-fold increase in cisplatin solubility overits room temperature solubility.
 12. The method of claim 11, whereinsaid adding includes adding a solution of vesicle-forming lipids heatedto within about 10° C. of the temperature of the cisplatin solution. 13.The method of claim 11, wherein said adding includes adding a solutioncontaining a vesicle-forming lipid having a phase transition temperaturewithin about 10° C. of the temperature to which the solution containingthe cisplatin compound is heated.
 14. The method of claim 10, whereinsaid adding includes adding a solution containing a vesicle-forminglipid having a phase transition temperature between 40-70° C.